Search for oncogene mutations in X-ray-transformed mouse 10T1/2 cells by denaturing gradient gel electrophoresis blotting.
Search for oncogene mutations in X-ray-transformed mouse 10T1/2 cells by denaturing gradient gel electrophoresis blotting.
复制标题
通过变性梯度凝胶电泳印迹寻找 X 射线转化的小鼠 10T1/2 细胞中的癌基因突变。
DOI：
10.1080/09553009414550181
复制
发表时间：
1994
影响因子：
2.6
通讯作者：
Little,JB
中科院分区：
文献类型：
--
作者：
Krolewski,B;Little,JB
我们用变性梯度凝胶电泳（DGGE）印迹技术寻找X射线转化的小鼠C3 H10 T1/2细胞克隆的c-myc、c-Ha-ras和c-Ki-ras基因座内可能突变的证据。开发这种高度灵敏的方法是为了检测基因组DNA中的任何突变（例如单碱基变化、小缺失），方法是测量短DNA片段（50-800 bp）的解链行为差异，该片段是通过用几种特定的4个碱基对识别位点进行消化而获得的。限制性酶。本研究将23个X射线转化的克隆的基因组DNA用几种限制性内切酶消化，变性梯度凝胶电泳，并与c-myc、c-Ha-ras和c-Ki-rascDNA探针杂交。与来自18个对照、非辐照野生型10 T1/2细胞克隆的DNA相比，未观察到这些癌基因中任何一个的熔解模式的改变，这表明转化与这些基因的突变或其甲基化模式的变化无关。然而，我们对c-mycas的外显子2和3以及c-Ha-ras基因的外显子1和2的大部分的筛选不能排除这样的可能性，即在所检查的片段的高解链结构域中的一些序列差异未被该测定法检测到。
We have sought evidence for possible mutations within the c-myc, c-Ha-rasand c-Ki-rasloci of X-ray-transformed mouse C3H10T½ cell clones using the denaturing gradient gel electrophoresis (DGGE) blot technique. This highly sensitive method was developed to detect any mutations (e.g. single base changes, small deletions) in genomic DNA, by measuring differences in the melting behaviour of short DNA fragments (50–800 bp) obtained by digestion of genomic DNA with several specific 4 bp recognition site restriction enzymes. In this study, genomic DNAs derived from 23 X-ray-transformed clones were digested with several restriction enzymes, electrophorezed on denaturing gradient gel and hybridized to c-myc, c-Ha-rasand c-Ki-rascDNA probes. No alterations in melting patterns were observed for any of these oncogenes as compared with DNA from 18 control, non-irradiated wild-type 10T½ cell clones, suggesting that transformation was not associated with mutation of these genes nor with changes in their patterns of methylation. However, our screening of the large portion of exons 2 and 3 of c-mycas well as of exons 1 and 2 of c-Ha-rasgene cannot exclude the possibility that some sequence differences in the high melting domains of examined fragments were not detected by this assay.